This invention relates generally to devices and methods for introducing a small quantity of a fluid sample into a sample vessel such as an ionization chamber, a capillary tube or a microfluidic device. More particularly, the invention relates to the use of nozzleless acoustic ejection to form and deliver droplets from a reservoir containing a small amount of fluid such as a microplate well, a capillary or a microfluidic device into a sample vessel for analysis and/or processing. The invention is particularly useful in mass spectrometry.
In the fields of genomics and proteomics, there is a need to manipulate, analyze and/or process minute quantities of sample materials. For example, microfluidic devices have been used as chemical analytic tools as well as means for introducing samples into clinical diagnostic tools. Their small channel size allows for the analysis of minute quantities of sample, which is an advantage where the sample is expensive or rare. However, microfluidic devices suffer from a number of unavoidable design limitations and drawbacks with respect to sample handling. For example, the flow characteristics of fluids in the small flow channels of a microfluidic device often differ from the flow characteristics of fluids in larger devices, as surface effects come to predominate and regions of bulk flow become proportionately smaller. Thus, in order to control sample flow, the surfaces of such devices must be adapted according to the particular sample to provide motive force to drive the sample through the devices. This means that a certain amount of sample waste must occur due to wetting of the device surfaces. Another limitation of microfluidic devices is the difficulty and expense of replicating the performance of some sophisticated chemical analytical instruments or chemical processing device within the microfluidic device. In these cases, it would be beneficial to have a method to remove a sample from the microfluidic device and load it directly into a more conventional analytical instrument or chemical reactor.
Surface wetting is a source of sample waste in other fluid delivery systems as well. For example, capillaries, Eppendorf-type or otherwise, having a small interior channel are often employed for sample fluid handling by submerging their tips into a pool of sample. In order to provide sufficient mechanical strength for handling, such capillaries must have a large wall thickness as compared to the interior channel diameter. Since any wetting of the exterior capillary surface results in sample waste, the high wall thickness to channel diameter ratio exacerbates sample waste. In addition, the sample pool has a minimum required volume driven not by the sample introduced into the capillary but rather by the need to immerse the large exterior dimension of the capillary. As a result, the sample volume required for capillary submersion may be more than an order of magnitude larger than the sample volume transferred into the capillary. Moreover, if more than one sample is introduced into a capillary, the previously immersed portions of the capillary surface must be washed between sample transfers in order to eliminate cross contamination. Cross contamination may result in a memory effect wherein spurious signals from a previous sample compromise data interpretation. In order to eliminate the memory effect, increased processing time is required to accommodate the washings between sample introductions.
Thus, there is a need in the art for techniques for manipulating minute amounts of fluids in conjunction with established analytical techniques. Mass spectrometry is a well-established analytical technique used for such analysis. In this technique, sample molecules are ionized and the resulting ions are sorted by mass-to-charge ratio. For sample molecules contained in a fluid sample, the sample fluid is typically converted into an aerosol that undergoes desolvation, vaporization, atomization, excitation, and ionization in order to form analyte ions.
For fluid samples, sample introduction is a critical factor that determines the performance of mass spectrometers, electrophoretic devices, and other analytical instruments. Analyzing the elemental constituents of a fluid sample may require the sample to be dispersed into a spray of small droplets or loaded in a predetermined quantity. A combination of a nebulizer and a spray chamber is commonly used for sample introduction, wherein the nebulizer produces the spray of droplets, and the droplets are then forced through a spray chamber and sorted by size and trajectory. Such droplets may be produced through a number of methods, such as those that employ ultrasonic energy and/or use a nebulizing gas. Such nebulizers, however, provide little control over the distribution of droplet size and no meaningful control over the trajectory of the droplets. As a result, the yield of droplets having a desired size and trajectory is low. In addition, the sample molecule may be adsorbed in the nebulizer, and large droplets may condense on the walls of the spray chamber. Such systems thus suffer from low analyte transport efficiency and high sample consumption.
Mass spectrometry has also been employed for samples that have been prepared as an array of features on a substrate. Matrix-Assisted Laser Desorption Ionization (MALDI), for example, is an ionization technique for large and/or labile biomolecules, such as nucleotidic and peptidic oligomers, polymers, and dendrimers, as well as for nonbiomolecular compounds such as fullerenes. MALDI is considered a xe2x80x9csoftxe2x80x9d ionizing technique in which both positive and negative ions are produced. The technique involves depositing a small volume of sample fluid containing an analyte on a substrate comprised of a photon-absorbing matrix material. The sample fluid typically contains a solvent and the analyte. Once solvent has been evaporated from the substrate, the analyte remains on the substrate at the location where the sample fluid is deposited. Photons from a laser strike the substrate at the location of the analyte and, as a result, ions and neutral molecules are desorbed from the substrate. Notably, the substrate matrix material is selected to provide enhanced desorption performance.
Surface Enhanced Laser Desoprtion Ionization (SELDI) is another example of a surface-based ionization technique that allows for high-throughput mass spectrometry. Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI employs a chemically reactive surface such as a xe2x80x9cprotein chipxe2x80x9d to interact with analytes, e.g., proteins, in solution. Such surfaces selectively interact with analytes and immobilize them thereon. Thus, analytes can be partially purified on the chip and then quickly analyzed in the mass spectrometer. By providing different reactive moieties at different sites on a substrate surface, throughput may be increased.
It should be evident, then, that sample preparation for surface-based ionization devices requires accurate and precise placement of carefully metered amounts of sample fluids on a substrate surface in order to reduce sample waste. Waste reduction is an important concern when sample fluids are expensive or difficult to obtain. In particular, certain biomolecular samples, e.g., nucleotidic and peptidic sample molecules, are exceptionally expensive. Thus, sample deposition on to a substrate often involves the use of small Eppendorf-type capillaries. These capillaries, of course, suffer from the disadvantages as discussed above.
Accordingly, it is desired to provide a device that requires only small volumes of sample to effect efficient sample delivery into analytical devices such as mass spectrometers and capillaries, that does not lead to compromised analysis due to the above-described memory effect, and that does not require long washing times.
A number of patents present different techniques for sample ionization and delivery. For example, U.S. Pat. No. 5,306,412 to Whitehouse et al. describes an apparatus that applies mechanical vibrations to an outlet port of an electrospray tip to enhance electrostatic dispersion of sample solutions into small, highly charged droplets, resulting in the production of ions of solute species for mass spectrometric analysis. The technology disclosed in this patent purports to overcome the problems associated with inkjet technology for sample ionization and delivery. The patent discloses that, due to plugging problems with nozzle orifices smaller than about 10 xcexcm, the techniques used in inkjet printing are not practical for the production of droplets in the size range required for efficient ion production in the mass spectrometric analysis of solutions. In addition, it is also disclosed that a single small orifice diameter associated with inkjet printers would not be effective over the flow rates associated with sample introduction in electrospray mass spectrometry. Like other electrospray systems, the described apparatus is disclosed to produce droplets of appropriate size but lacks control over the trajectory of droplets as they depart from the electrospray tip.
A number of patents relate to the use of acoustic energy in printing. For example, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquid drop emitter that utilizes acoustic principles for ejecting liquid from a body of liquid onto a moving document for forming characters or bar codes thereon. Lovelady et al. is directed to a nozzleless inkjet printing apparatus wherein controlled drops of ink are propelled by an acoustical force produced by a curved transducer at or below the surface of the ink. In contrast to inkjet printing devices, nozzleless fluid ejection devices as described in the aforementioned patent are not subject to clogging and the disadvantages associated therewith, e.g., misdirected fluid or improperly sized droplets. In other words, nozzleless fluid delivery provides high fluid-delivery efficiency through accurate and precise droplet placement. Nozzleless fluid ejection also provides a high level of control over ejected droplet size.
While nozzleless fluid ejection has generally been limited to ink printing applications, it is not completely unknown in the field of ionized fluid delivery. U.S. Pat. Nos. 5,520,715 and 5,722,479, each to Oeftering, describe an apparatus for manufacturing a freestanding solid metal part through acoustic ejection of charged molten metal droplets. The apparatus employs electric fields to direct the charged droplets to predetermined points on a target where the droplets are solidified as a result of cooling. It should be readily evident that the apparatus disclosed in these patents employs acoustic ejection for metallic part synthesis rather than for biomolecular analysis. In addition, a high temperature is required in order to melt most metal samples, and such an apparatus would be incompatible with samples that decompose or are otherwise adversely affected by exposure to high temperatures.
Thus, there is a need in the art for improved sample preparation and introduction that employs acoustic ejection to deliver a small quantity of a fluid sample with accuracy, precision, and efficiency.
Accordingly, it is an object of the present invention to provide devices and methods that overcome the above-mentioned disadvantages of the prior art. One embodiment of the invention relates to a device having a sample vessel for processing and/or analyzing a sample molecule. The analytical device also includes an acoustic ejector for introducing the sample molecule from a reservoir holding a fluid sample comprised of sample molecule. The ejector comprises an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation at a focal point near the surface of the fluid sample. Furthermore, a means for positioning the ejector in acoustic coupling relationship to the reservoir is provided. When the sample vessel is an ionization chamber, the analytical device, for example, may be a time-of-flight mass spectrometer that allows analysis of various types of sample molecules, such as biomolecules having a high molecular weight. In addition, the sample vessel may either comprise a microfluidic device or represent a portion of a microfluidic device.
Typically, the inventive devices allow for droplet ejection from a small volume of fluid. For instance, the fluid sample may occupy a volume in the picoliter range, and the ejected droplets may occupy a volume in the femtoliter range. Moreover, acoustic ejection allows precise and accurate control over droplet trajectory.
Another embodiment of the invention relates to a method for introducing a sample molecule into a sample vessel of a device for processing an/or analyzing a sample molecule. The inventive method provides for a reservoir holding a fluid sample comprised of the sample molecule and employs focused acoustic radiation directed at a point near the surface of the fluid sample to eject a droplet of the fluid sample therefrom along a predetermined trajectory into the sample vessel of the device. The method allows for accuracy and precision in the formation and placement of ejected droplets such that the ejected droplets may be substantially identical in size and follow substantially identical trajectories. As before, the sample vessel may be an ionization chamber or a microfluidic device.
Still another embodiment of the invention relates to an analytical device having an sample vessel for processing and/or analyzing a plurality of sample molecules. The device includes a plurality of reservoirs each holding a fluid sample comprised of a sample molecule, an ejector comprising an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation at a focal point near the surface of the fluid sample, and a means for positioning the ejector in acoustic coupling relationship to each of the reservoirs to eject a droplet of fluid sample into the sample vessel. In some cases, the reservoirs are arranged in an array, such as in the case where the reservoirs comprise designated sites on a single flat substrate surface. Preferably, a means for altering the spatial relationship of at least one reservoir with respect to the ionization chamber is also provided.
In a further embodiment, the invention relates to a method for introducing fluid samples into a sample vessel of a device of processing and/or analyzing a sample molecule. The method involves: (a) providing a plurality of reservoirs each holding a fluid sample having a fluid surface; (b) positioning an ejector in acoustically coupled relationship to a selected reservoir; (c) activating the ejector to generate acoustic radiation having a focal point near the fluid surface of the fluid sample held in the selected reservoir to eject a droplet of fluid sample into the sample vessel; and (d) optionally repeating steps (b) and (c) for an additional reservoir. Typically, the method allows for locating the fluid surface of the fluid sample held by the selected reservoir before ejecting one or more droplets from the reservoir. The surface of the fluid sample may be located by detecting for reflected acoustic radiation from the fluid sample. Optionally, acoustic reflections may be used to align the acoustic focus with the opening of the ionization chamber or capillary.
Thus, the invention also provides a method for preparing a plurality of sample molecules for analysis. Such preparation involves applying focused acoustic energy to each of a plurality of fluid-holding reservoirs, each of said reservoirs holding sample molecules in a fluid to be applied to a designated site on the substrate surface in order to prepare an array comprised of a plurality of sample molecules on a substrate surface. Once the array is prepared, sufficient energy is successively applied to each site to ionize the sample molecules and release the sample molecules from the substrate surface for analysis. The energy may be applied, e.g., by bombarding the sites with acoustic energy, photons, electrons, and/or ions.
Furthermore, the invention provides a method for preparing a sample surface for analysis. The method involves first placing a sample surface in droplet-receiving relationship to a fluid-holding reservoir holding an analysis-enhancing fluid. Then, focused acoustic energy is applied in a manner effective to eject a droplet of the analysis-enhancing fluid from the reservoir such that the droplet is deposited on the sample surface at a designated site. Finally, the sample is subjected to conditions sufficient to allow the analysis-enhancing fluid to interact with the sample surface to render the substrate surface at the designated site suitable for analysis. Optionally, sufficient energy is applied to the designated site to ionize the sample surface and to release sample molecules from the substrate surface for analysis.
In another embodiment, the invention relates to a device useful for the efficient transport of a fluid sample. The device comprises a sample vessel having an inlet opening with a limiting dimension of no more than about 300 xcexcm, a reservoir holding a fluid sample having a volume of no more than about 5 xcexcL, and an ejector configured to eject at least about 25% of the fluid sample through the inlet opening into the sample vessel. Typically, the ejector comprises an acoustic radiation generator for generating radiation, a focusing means for directing the radiation at a focal point near the surface of the fluid sample, and a means for positioning the ejector in acoustic coupling relationship to the reservoir. Optionally, the ejector does not directly contact the radiation generator. The efficiency of this device lies in the ability of the device to handle extremely small fluid samples with little or no sample waste. Similarly, another embodiment of the invention relates to a method for the efficient transport of a droplet of a fluid sample, wherein a reservoir is provided holding a fluid sample having a volume of no more than about 5 xcexcL, and at least 25% of the fluid sample is ejected through an inlet opening of a sample vessel, the inlet opening having a limiting dimension of no more than about 300 xcexcm.
In a still further embodiment, a device for efficient transport of a fluid sample is provided. The device comprises: a sample vessel having an inlet opening with a limiting dimension of no more than about 10 xcexcm to about 300 xcexcm; a reservoir holding a fluid sample having a depth of about 0.1 to about 30 times the limiting dimension of the inlet opening; and an ejector configured to eject a droplet of the fluid sample through the inlet opening into the sample vessel. The sample vessel of the device does not contact the fluid sample held by the reservoir.
In yet another embodiment, a device for efficient transport of a fluid sample is provided, comprising: a sample vessel having an inlet opening with a limiting dimension; a reservoir holding a fluid sample having a depth of about 0.1 to about 30 times the limiting dimension of the inlet opening; and an acoustic ejector. The acoustic ejector is configured to eject a droplet of the fluid sample through the inlet opening into the sample vessel and comprises an acoustic radiation generator for generating acoustic radiation having a predetermined wavelength in the fluid sample selected according to the limiting dimension of the inlet opening of the sample vessel or the depth of the fluid sample and a focusing means for focusing the acoustic radiation at a focal point near the surface of the fluid sample, wherein the acoustic ejector is in acoustic coupling relationship to the reservoir.
In a further embodiment, the invention relate to an apparatus for ejecting a fluid droplet from a microfluidic device. A microfluidic device is provided comprising a base having a microchannel formed in a surface thereof, and a cover plate arranged over the base surface. The cover plate in combination with the microchannel defines a microconduit, and the microconduit fluidly communicates with an inlet opening and an outlet opening. An ejector is also provided comprising an acoustic radiation generator for generating acoustic radiation, and a focusing means for focusing the acoustic radiation at a focal point near the surface of a fluid at the outlet opening of the microfluidic device. A positioning means serves to position the ejector in acoustic coupling relationship to the microfluidic device to eject a droplet from the outlet opening of the microfluidic device.
In still another embodiment, a method is provided for ejecting a fluid droplet from a microfluidic device. A microfluidic device is provided comprising a base having a microchannel formed in a surface thereof, and a cover plate arranged over the base surface. As above, the cover plate in combination with the microchannel defines a microconduit, and the microconduit fluidly communicates with an inlet opening and an outlet opening. When focused acoustic radiation is directed at a point near the surface of a fluid at the outlet opening of the microfluidic device, a droplet of the fluid is ejected from the outlet opening of the microfluidic device.